1. Field of the Invention
The present invention is generally directed to LC/MS analysis of high molecular weight proteins, including antibodies and antibody conjugates or fragments.
2. Background of the Related Art
Purification techniques such as high performance liquid chromatography (HPLC) that are readily adaptable for the separation and analysis of low molecular weight species often have limited application when being used to separate protein species larger than 50 kDa. In addition, the characterization and/or consistency of manufacture of a given protein often relies on peptide mapping in order to monitor the amino acid sequence and/or conformational properties of the protein being analyzed. While peptide mapping and analysis of intact proteins are able to detect small changes in small- to moderate-sized proteins, for example, insulin (molecular weight 6 kDa) and human hormone of molecular weight 22 kDa (Oroszlan et al., Anal. Chem., v. 64, p. 1623-1631, 1992), this kind of detailed analysis of a larger protein, for example, heterogeneous glycoproteins such as antibodies (150 kDa) requires cleavage of these proteins into a large number of peptides. The analysis of these larger proteins by peptide mapping is thus, hindered by the complexity of the range of peptides generated by timely enzymatic digestion or non-specific catalytic or hydrolytic digestion of the protein and separation of the multiple peptides. Therefore, reversed-phase (RP) HPLC of large proteins is an attractive alternative approach to peptide mapping, because the former method often does not require any sample preparation and is relatively simple in data interpretation.
In 1984 (Geng and Regnier, J. Chromatogr., v. 296, p. 15-30, 1984), a retention model was proposed for proteins in reversed-phase HPLC incorporating three processes: adsorption, solvation and desorption. According to the model, protein elution is a result of a stoichiometric displacement of protein on the alkyl-bonded surface by molecules of organic solvent. In that study, iso-propanol was identified as a stronger displacing agent as compared to methanol and ethanol. The paper also suggested that change of chromatographic retention with temperature is governed by altering both the forces of interaction between molecular species (the forces of attraction between all the components decrease with increasing temperature) and the protein conformation (Geng and Regnier, J. Chromatogr., v. 296, p. 15-30, 1984).
Other efforts to improve RP-HPLC separation of large proteins included optimization of the n-alkyl silica phase, mobile phase, column temperature and pore size. Nevertheless, there remains a significant problem in RP chromatography of large proteins in that a single protein generally produces broad, asymmetrical and even multiple chromatographic peaks at near-room temperatures. These phenomena have previously been documented in several reports (Cohen et al., Anal. Biochem., v. 140, p. 223-235, 1984a; Cohen et al., Anal. Chem., v. 56, p. 217-221, 1984b,; Hearn et al., J. Chromatogr., v. 435, p. 271-284, 1988; Lu et al., J. Chromatogr. A, v. 359, p. 19-29, 1986; Oroszlan et al., Anal. Chem., v. 64, p. 1623-1631, 1992; Purcell et al., Anal. Chem., v. 71, p. 2440-2451 1999a; Richards et al., J Chromatogr. A, v. 676, p. 33-41, 1994; Richards et al., J. Chromatogr. A, v. 676, p. 17-31, 1994) and may be related to partially unfolded conformational intermediates trapped on the stationary phase under gradient elution conditions.
Mass spectrometry is a powerful technique for characterization of proteins including large proteins. A mass spectrometer includes two main components: 1) ion source, which desorbs protein molecules from liquid or solid state into the gas phase and ionizes them and 2) mass analyzer, which determines the molecular weight values of the protein ions. Electrospray ionization (ESI), (Fenn et al., Science, 246, 64, 1989) and matrix-assisted laser desorption/ionization (MALDI), Karas and Hillenkamp, Anal. Chem. 60, 2299 (1988); Tanaka et al., Rapid Communications in Mass Spectrometry, 2, 151-153 (1988), are the two most powerful modern ion sources for protein analysis by mass spectrometry. In these sources, a positively charged protein ion is formed by attaching one or several protons to the molecule of protein. Ion trap (IT), quadrupole (O), magnetic sector, Fourier transform ion cyclotron resonance (FT-ICR), time-of-flight (TOF) and orthogonal time-of-flight (orthogonal TOF) are the mass analyzers typically employed for proteins analysis. These analyzers produce mass spectrum with m/z values of detected ions on x-axis and ion intensity on y-axis. MALDI typically produces ions with low charge. For example, a 150 kDa (m) antibody typically produces ions with +1, +2 and +3 charges (z) and m/z values of 150 kDa, 75 kDa and 50 kDa, correspondently, Akashi et al., Anal. Chem. 70(15):3333-6, 1998; Alexander and Hughes, Anal. Chem. 67 (20):3626-32, 1995.
The TOF and orthogonal-TOF analyzers posses the largest m/z scale and most suitable for the large protein analysis. It was found that detection efficiency of the protein ions decreases with increasing their m/z values. The ESI is more efficient ionization technique, which produces multiply charged ions with large number of charges (z) significantly reducing their m/z range and improving their detection efficiency. Typically, larger proteins will produce ions with larger m/z values. For example, one of the advantages of ESI source as compared to MALDI is that the former is suitable for in-line operation with HPLC. In the ESI source, the proteins eluting from an HPLC column are ionized and ejected into the gas at atmospheric pressure. Then the ions are guided into the vacuum through an atmosphere-vacuum interface into the mass analyzer. The orthogonal TOF analyzers were found to be a good choice for the LC/MS analysis, because it transforms the continuous flow of ions from the ESI source into the pulsed beam required for TOF analysis using an orthogonal accelerator Dodonov et al., Rapid Comm. Mass Spec. 11(15):1649-56, 1997; Guilhaus et al., Mass Spec. Rev. 19(2):65-107, 2000. Taking into account the above considerations, the ESI orthogonal TOF mass spectrometer is currently the best choice for LC/MS analysis of large proteins. The other mass analyzers, such as the listed above (IT, Q, FT-ICR, magnetic sector) are also applicable after appropriate tuning.
The discovery that multiply charged ions produced by electrospray mass spectrometry (ESI-MS) can be deconvoluted to determine the molecular mass of a protein with masses in excess of the conventional m/z range of a mass spectrometric analyzer has led to the use of ESI-MS for the analysis of protein structures (see discussion in Fenn et al., Science 246, 64, 1989). ESI-MS has been successfully used to analyze proteins having a MW of approximately 50 kDa (see Whitelegge et al., Protein Sci. 1998 June; 7(6):1423-30, 1998, presenting analysis of intact membrane proteins of MW of 42 kDa). While mass spectrometry has been used for the analysis of small protein fragments of less than 90 kDa, the applicability of mass spectrometry for the analysis of higher molecular weight proteins has not been effectively achieved.
In addition to the use of HPLC and MS separately, commercially available combined HPLC and electrospray ionization mass spectrometry (LC-ESI-MS) systems compatible with conventional HPLC has proven useful in peptide mapping (Ling et al., Anal. Chem., 63: 2909-2915, 1991; Guzzetta et al., Anal. Chem., 65: 2953-2962, 1993; Bongers et al., J. Pharm. Biomed. Anal., v. 21, p. 1099-1128., 2000). LC-ESI-MS in combination with in-source collisionally induced dissociation (CID) has been used effectively to identify sites of N- and O-linked glycosylation, but again this methodology is only effective with peptides or small protein fragments (Carr et al., Protein Sci., 2: 183-196, 1993; Huddleston et al., Anal. Chem., 65: 877-884, 1993; Conboy and Henion, J. Am. Soc. Mass Spectrom., 3: 804-814, 1992). However, these techniques remain inadequate for analysis of antibodies and other large conformationally complex proteins. This is due to an insufficient resolution resulting from the confounding effects of the large numbers of very similar peptides that result from variable protein glycosylation and enzymatic digests of moderately sized glycoproteins. It is therefore necessary to employ a range of techniques with orthogonal selectivity to characterize such samples.
The use of combinations of high-performance capillary electrophoresis, HPLC, LC-ESI-MS, and matrix-assisted laser desorption ionization-time of flight mass spectrometry has been investigated to allow for characterization of enzymatic digests of underivatized glycoprotein samples, as exemplified by DSPα1, a single-chain plasminogen activator derived from vampire bat salivary glands (Apffel et al, J. Chromatography A, 717: 41-60, 1995). It was concluded that these four techniques when used in combination are complimentary techniques for examining glycoproteins, although only on protein digests rather than on high molecular weight proteins. Nonetheless, the authors acknowledged that more work needs to be done to improve the power of this approach, and that high-yield concentration steps will be required due to extensive carbohydrate heterogeneity.
Thus, useful information from the LC-ESI-MS or MALDI-TOF techniques can only be obtained from analysis of intact proteins that are relatively small, e.g., less than 10 kDa. Although the peptide mapping can be used to obtain the more detailed information, this method consumes a lot of time for sample preparation and data interpretation and becomes very complicated when molecular weight of protein increases. However, many biologically-relevant proteins have higher molecular weights and further, have post-translational modifications that can confound their analyses in mass spectrometry and/or HPLC. Thus, despite the fact that there are techniques that have been extensively used in the analysis of low molecular weight proteins such as insulin, or low molecular weight digests of larger proteins, there remains a need for additional methods and techniques for producing sequence and detailed conformational information about proteins.